In Drosophila, arguably the best-understood multicellular organism and a proven model system for human diseases, less than half of the 15,185 annotated genes have mutations, and many fewer have readily detectable phenotypes. Our lack of functional information on the majority of the genes, also referred to as the "Phenotype Gap", does not indicate that these genes have no function but rather that, as experimentalists, we have been unable to either assay their roles or resolve the issue of functional redundancy. Conditional expression of hairpin constructs in Drosophila has emerged in recent years as the method of choice to fill in the "Phenotype Gap", as well as to overcome the issues associated with gene pleiotropy. Using transgenic RNAi it is now possible to disrupt the activity of single genes with a spatial and temporal resolution that is impossible or exceedingly difficult using classical genetic methods. Here, we propose to build a resource of 6,250 transgenic RNAi lines, the "Transgenic RNAi Resource Project", using improved methodology developed in our laboratory. The lines will be established and validated at the Drosophila RNAi Screening Center (DRSC) and transferred to the Bloomington Drosophila Stock Center (BDSC) to be made freely available to the community. We anticipate this resource to be built, tested and transferred in its entirety to the BDSC over a four-year period. This collection of transgenic RNAi lines will be invaluable to address a myriad of questions in biology and medicine, including, but not limited to, cell biology, signal transduction and cancer, the etiology of congenital malformations, neurodegenerative studies, and behavior.